Kinetics of cellular cytotoxicity mediated by a cloned human natural killer cell line

The kinetics of natural killer (NK) cytotoxicity mediated by the cloned interleukin 2 (IL 2)-dependent human natural killer cell line NK3.3 has been investigated. This cloned cell line exhibits strong cytotoxic activity that is restricted to NK target cells. The initial rate of lysis of K-562 target cells by these cloned NK cells was, as anticipated, substantially greater than that previously reported for human peripheral blood NK cell preparations. However, in contrast to the kinetics of NK cytolysis mediated by fresh peripheral NK cells, the rate of 51Cr-release declined substantially within 1 to 3 h after initiation of assays involving NK3.3 cells and reached a plateau value in experiments conducted for longer periods. The data obtained suggest that NK3.3 cells cannot readily recycle and kill multiple target cells. Based on a model involving one lethal hit per active NK3.3 cell, estimates for the frequency of cytolytic cells in NK3.3 cultures were computed and compared to estimates obtained by the application of a kinetic model previously described. The cytotoxic activity of the NK3.3 cells was also found to be highly dependent on the conditions used for propagation and assay of these cells and, when cultured under "standard" conditions, only a fraction of the cloned NK3.3 cells was capable of effecting lysis of K-562 target cells. However, for the most optimal conditions developed to date, each NK cell killed an average of 1 to 2 target cells before inactivation. Although no significant differences in viability or growth rate were observed for the three different conditions described, up to 300-fold differences in lytic activity were observed. The observed strong dependence of the lytic function of NK3.3 cells on culture conditions should prove valuable for further investigations of mechanisms governing the regulation and function of human NK cells.     

CD28-dependent killing by human YT cells requires phosphatidylinositol 3-kinase activation

CD28/B7 interactions have been demonstrated to provide a co-stimulatory signal for the generation of CD8⁺ cytotoxic T lymphocytes in the absence of CD4⁺ T helper cells. The CD28 signals required for induction of cytotoxicity have yet to be described. To investigate further the biochemical signaling pathways associated with CD28-dependent cytotoxicity, we have studied the human thymic leukemia cell line, YT. YT cells kill B7⁺ targets in a non-major histocompatibility complex (MHC)-restricted, CD28-dependent manner. CD28 ligation on the surface of YT cells caused a rapid increase in the tyrosine phosphorylation of four major cellular substrates with masses estimated to be 110, 95, 85, and 44 kDa. The 110 and 85 kDa substrates were identified as the catalytic and regulatory subunits, respectively, of phosphatidylinositol 3-kinase (PI3-K). Engagement of CD28 caused the rapid receptor association and activation of PI3-K but did not activate phospholipase C_γ. CD28-induced tyrosine phosphorylation and PI3-K activation was independent of p56^lck protein tyrosine kinase (PTK) activity (previously reported to be associated with CD28) and was insensitive to inhibition by the PTK inhibitor herbimycin A. Two structurally and mechanistically dissimilar inhibitors of PI3-K, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) also failed to block CD28-dependent tyrosine phosphorylation events or the association of PI3-K with the CD28 receptor. However, both drugs inhibited CD28-dependent cytotoxicity and CD28 receptor associated PI3-K activity with IC₅₀ values similar to the reported IC₅₀ values for PI3-K inhibition. Although herbimycin A did not significantly block the observed CD28-dependent tyrosine phosphorylation or PI3-K activation, herbimycin did block CD28-dependent cytotoxicity in a dose-dependent manner. These data support a role for PI3-K activation in the CD28-dependent initiation of cytotoxic effector function and suggest that a herbimycin sensitive step(s) is either CD28-independent, resides within a PI3-K-independent CD28 signaling pathway, or is downstream of CD28-dependent PI3-K activation.     

Inhibition of T cell apoptosis by IFN-beta rapidly reverses nuclear translocation of protein kinase C-delta.

Type I interferons rescue activated human T cells from cytokine deprivation-induced apoptosis. Our data now show that IFN-beta also rapidly inhibits apoptotic signals induced through the Fas receptor (CD95) in human T cells. To identify upstream signaling elements that could be targets of IFN-beta, we have studied protein kinase C (PKC). PKC-delta is actively involved in the regulation of apoptosis and immunofluorescence staining revealed that early in apoptosis PKC-delta accumulated in the nucleus. Addition of IFN-beta to T cells already deprived of survival factors or treated with anti-Fas antibody caused a rapid retranslocation of PKC-delta away from the nucleus. Furthermore, the generation of a constitutively active catalytic fragment by cleavage of PKC-delta by caspase 3 occurred only after translocation of full-length PKC-delta to the nucleus. IFN-beta also inhibited caspase 3 and the proteolytic activation of PKC-delta. We conclude from these studies that nuclear translocation of PKC-delta is an early event in T cell apoptosis and that IFN-beta rapidly reverses this process.     

Ligation of the T cell co-stimulatory receptor CD28 activates the serine-threonine protein kinase protein kinase B

The intracellular signaling pathways activated upon ligation of the co-stimulatory receptor CD28 remain relatively ill-defined, although CD28 ligation does result in the strong association with, and activation of, phosphatidylinositol (PI) 3-kinase. The downstream effector targets of the CD28-activated PI 3-kinase-dependent signaling pathway remain poorly defined, but recent evidence from other systems has shown that Akt/protein kinase B (PKB) is a major target of PI 3-kinase and have indicated that a major function of PKB is the regulation of cell survival events. Given the strong coupling of CD28 to PI 3-kinase and the known protective effects of both CD28 and PI 3-kinase against apoptosis in different cell models, we investigated the effects of CD28 on PKB activation. We demonstrate that ligation of CD28 by either anti-CD28 monoclonal antibodies or the natural ligand B7.1, results in the marked activation of PKB in both the leukemic T cell line Jurkat and freshly isolated human peripheral blood-derived normal T lymphocytes. Our data suggest therefore, that PKB may be an important intracellular signal involved in CD28 signal transduction and demonstrate CD28 coupling to downstream elements of a signaling cascade known to promote cell survival.     

Regulation of non-classical protein kinase C isoenzymes in a human T cell line

We have examined the expression and responses to activation, of novel/atypical protein kinase C (PKC) isoforms ε,ζ, and δ in the T cell lymphoma cell line K-4. The effects of 1-h phorbol 12-myristate 13-acetate (PMA) and OKT3 activation of K-4 cells on PKC isoform distribution were examined. In addition, the effects of PMA-mediated down-regulation on the expression of PKC ε and ζ were determined using high concentrations of PMA over 24- and 48-h time periods in these cells. PKC ε expression was not altered by incubation of K-4 cells with up to 200 ng/ml PMA over a 24- or 48-h period. PKC ε was down-regulated in a concentration-dependent manner by PMA after both 24- and 48-h of activation. Expression of PKC ε was not completely depressed, however, even at the highest concentration of the phorbol ester after 48-h incubation with PMA. The presence of PKC ε, ζ, and δ was confirmed by immunohistochemistry with distinct patterns of expression observed. PMA-induced PKC activation for a 1-h period resulted in a translocation of PKC δ from resting cytoplasmic/nuclear staining to a cytoplasmic aggregate. Following 1-h activation through the T cell receptor-associated complex CD3, PKC δ translocated from a peri-nuclear/cytoplasmic compartment to a putative cytoskeletal location in K-4 cells. This translocation was time dependent and redistributed to a cytoplasmic aggregate prior to the cytoskeleton. Similarily, following 1-h activation through the T cell receptor, PKC ζ redistributed directly to what is possibly a cytoskeletal cell compartment. The cytoplasmic distribution of PKC ζ was unaltered following activation with PMA over a 1-h time period. There was no apparent redistribution of PKC ε cytoplasmic staining pattern following a 1-h direct or indirect activation. These results underline the differences in individual PKC isoform distribution, and responses to different stimuli, thereby providing additional evidence for the use of discrete PKC isoform signaling pathways in T cells. Furthermore, this data underlines the differences in PMA-mediated PKC activation and activation through the T cell receptor.     

Antibody-dependent cytotoxicity on chicken red blood cell targets mediated by the U937 cell line.

We have characterized a model system for the study of antibody-dependent cytotoxicity (ADCC) mediated by human macrophages and monocytes. The U937 cell line is used as a source of effector cells. We confirmed a previous report (Gidlund et al., 1981) that U937 can be activated using PMA to kill in ADCC, and the characteristics of the observed cytotoxicity are described. Activation of effectors was maximal after 20-hr preincubation in the presence of 10 ng/ml PMA. In these conditions, lysis was approximately 70% in 2 hr at an effector to target ratio of 5:1. Activation correlated with the expression of complement receptors CR1 and CR3. No antibody-independent cytotoxicity was observed. The effects of various inhibitors of oxygen species were investigated: the lysis obtained in the above conditions was inhibited at 50% in the presence of either 450 mM dimethyl sulphoxide or 30,000 U/ml catalase, but superoxide dismutase (up to 10,000 U/ml) or ferricytochrome c (up to 2 mM) had no effect. The same inhibition was observed with 40 mM desferrioxamine or with 1 mM 0-phenanthroline, which are both iron scavengers, or in the presence of 300 microM colchicine or 1.5 microM dihydrocytochalasin B, which are two inhibitors of cytoskeletal functions. An identical effect was obtained in the presence of 1 TIU/ml bovine pancreas trypsin inhibitor, whereas soya bean trypsin inhibitor, which is more specific, had no effect up to 5000 BAEE U/ml. No inhibition was seen with protein synthesis inhibitors as cycloheximide or puromycin at 40 micrograms/ml. The significance of these results is discussed.     

Tau-ubiquitin protein conjugates in a human cell line.

The microtubule-associated protein tau, and the cytoplasmic protein ubiquitin, are constituents of pathological neurofibrillary tangles found in Alzheimer's disease. In order to see if there is any physiological relationship between these proteins in a functioning human system, human neuroblastoma (LAN-5) cells were grown in vitro and differentiated to a neuronal phenotype. Cell extracts were analyzed by SDS-PAGE, immunoblot, and immunoprecipitation techniques. The colocalization of ubiquitin and tau immunoreactivity was noted in 12- and 35-kDa bands, predominantly located in a cell membrane fraction. The bands were also isolated by immunoprecipitation with the Alz-50 antibody and then identified with a ubiquitin antiserum. These findings show a relationship between tau and ubiquitin in a human neural cell line. This interaction suggests that tau may normally be degraded by an ubiquitin-dependent mechanism and alterations in it may contribute to the formation of neuro-fibrillary pathology.     

Overexpression of protein kinase Cdelta enhances cisplatin-induced cytotoxicity correlated with p53 in gastric cancer cell line.

OBJECTIVE: An important issue in cancer therapy is to investigate the mechanism for cellular sensitivity to anticancer agents such as cisplatin. Cisplatin is one of the DNA-damaging agents and several factors including p53 are related to the sensitivity to cisplatin in cancer. Protein kinase C (PKC) delta is known as a positive regulator for cisplatin-induced cell death. In our present study, we examined whether overexpression of PKCdelta and p53 increases the sensitivity of the human gastric cancer cell line, MKN28, which has a mutation of p53 gene, to cisplatin. METHODS: Cell viability and DNA content were measured in MKN28 with adenovirus-mediated expression of PKCdelta and p53 after exposure to cisplatin. In addition, the active form of caspase-3 was detected by Western blotting. RESULTS: Overexpression of exogenous PKCdelta did not induce cell death in MKN28 but inhibited cell growth at 1 microg/ml cisplatin as compared to that by cisplatin alone. Moreover, overexpression of both wild-type p53 and exogenous PKCdelta in MKN28 increased cisplatin-induced cell death in MKN28. CONCLUSION: These results suggest that PKCdelta, in cooperation with p53, possibly regulates cisplatin-induced caspase-3-mediated cell death in gastric cancer.     

The role of lymphocyte subsets and adhesion molecules in T cell-dependent cytotoxicity mediated by CD3 and CD28 bispecific monoclonal antibodies

The cure of human Hodgkin's tumors heterotransplanted into SCID mice can be achieved by two bispecific monoclonal antibodies (Bi-mAb) directed against the tumor-associated CD30 antigen and CD3 and CD28, respectively, and normal peripheral human blood T cells. We investigated the role of lymphocyte subsets and adhesion molecules in this Bi-mAb-mediated cytolysis. CD4⁺ lymphocytes were the most rapidly expanding subpopulation, but Bi-mAb-directed cytotoxicity was mediated preferentially by CD8⁺ lymphocytes and effector cells belonging to the CD45RO⁺ “memory” pool. Blocking of the LFA-1/ICAM-1 or CD2/LFA-3 adhesion pathways by mAb decreased Bi-mAb-mediated cytotoxicity. This was not due to inhibition of aggregate formation between Bi-mAb-coated T lymphocytes and target cells. Cross-linking of LFA-1 or CD2 molecules on lymphocytes prestimulated with Bi-mAb bound to CD3 and CD28 antigen lead to a more pronounced and prolonged rise in the intracellular concentration of free Ca²⁺. Additional CD2 cross-linking resulted in the tyrosine phosphorylation of distinct proteins. These findings indicate that adhesion molecules play a critical role and function as co-stimulatory signals rather than as cellular contact mediators in CD3 and CD28 Bi-mAb-stimulated T lymphocytes.     

Contractile potentiation by endothelin-1 involves protein kinase C-delta activity in the porcine coronary artery.

The relationship between potentiation of serotonin (5-hydroxytryptamine, 5-HT)-induced contraction by endothelin-1 (ET-1) and the activity of protein kinase C (PKC) was examined in the porcine coronary artery. Low concentrations (30-100 pM) of ET-1 selectively potentiated the 5-HT-induced contraction 1.5 to 2 times over the control without any additional increase in myosin light-chain phosphorylation. The potentiation was attenuated by PKC down-regulation caused by phorbol 12-myristate 13-acetate. By Western blot analysis with isoform-specific antibodies to PKC, at least four isoforms (PKCalpha, PKCbeta1, PKCdelta and PKCzeta) were identified in the porcine coronary artery. PKCalpha and PKCdelta were mostly in the cytosol fraction, whereas PKCbeta1 and PKCzeta were almost equally distributed in the cytosol and membrane fractions in the resting and contractile states. Of the four isoforms, only PKCdelta was translocated from the cytosol to the membrane fraction during the contractile potentiation by ET-1. These results suggest that the activity of PKCdelta, a Ca2+-independent PKC isoform, is involved in the potentiation of 5-HT-induced contraction by ET-1 in the porcine coronary artery.     

Rottlerin-independent attenuation of pervanadate-induced tyrosine phosphorylation events by protein kinase C-delta in hemopoietic cells

The understanding and control of many pathophysiological conditions is based on knowledge of subtly regulated intracellular signaling networks. We have found that in pervanadate (PV)-treated J558L myeloma cells, amongst other signaling proteins, protein kinase C (PKC)-delta and src homology 2-containing inositol phosphatase (SHIP) are tyrosine phosphorylated on expression of the B cell receptor, suggesting a role for these proteins in the preformed B cell receptor transducer complex. Rottlerin, a widely used PKC-delta-specific inhibitor, efficiently blocks these PV-induced tyrosine phosphorylation events. Furthermore, PV treatment of bone marrow-derived mast cells (BMMC) also results in tyrosine phosphorylation of PKC-delta, SHIP, and additional proteins. Rottlerin also inhibits these responses, indicating that PKC-delta might play an important enhancing role in the propagation of phosphotyrosine signals in B cells and mast cells and hence in the regulation of function of both cell types. Therefore, BMMC from PKC-delta -/- mice were generated by in vitro differentiation and assayed for tyrosine phosphorylation events in response to PV. Intriguingly, and opposite to the Rottlerin data, PKC-delta -/- BMMC show a stronger response to PV than wild-type cells, suggesting an attenuating role for PKC-delta. This response can be inhibited equally well by Rottlerin, indicating clearly that Rottlerin is not specific for PKC-delta in vivo. A comparison between Rottlerin and the panspecific PKC inhibitor bisindolylmaleimide suggests that Rottlerin also targets kinases beyond the PKC family. Moreover, Ser473 phosphorylation of protein kinase B (PKB) after PV treatment is blocked by Rottlerin as efficiently as by the phosphatidylinositol 3-kinase inhibitor LY294002. In this report, we provide evidence that PKC-delta constitutes a crucial attenuating factor in B cell and mast cell signal transduction and suggest that PKC-delta is important for the regulation of physiological B and mast cell functions as well as for their pathophysiology. Furthermore, dominant PKC-delta-independent effects of Rottlerin are presented, indicating restrictions of this inhibitor for use in signal transduction research.     

Inhibition of CD28-mediated natural cytotoxicity by KIR2DL2 does not require p56(lck) in the NK cell line YT-Indy.

CD28 functions as a cytotoxicity activation receptor in the NK cell line YT-Indy. To analyze the requirement of p56(lck) kinase in the function of killer inhibitory receptors, we transfected the p56(lck) negative YT-Indy cell line with the cl43 gene encoding for KIR2DL2. Pervanadate treatment revealed KIR2DL2 phosphorylation in YT-Indy-cl43, as well as SHP1/SHP2 recruitment. YT-Indy-cl43 cells were inhibited in their ability to lyse target cells expressing HLA-Cw3, a ligand for KIR2DL2. This inhibition was blocked by anti-KIR2DL2 or anti-HLA class I mAb. CD28 crosslinking on YT-Indy-cl43 enhanced tyrosine phosphorylation of PLC-gamma1. The simultaneous ligation of KIR2DL2 with mAb resulted in a decrease in CD28-induced tyrosine phosphorylation of PLC-gamma1 confirming that dephosphorylation of this protein is involved in the KIR2DL2-induced inhibition of CD28-mediated cytotoxicity. As YT-Indy-cl43 did not express detectable levels of p56(lck), these results indicate that this kinase is not required for transmitting the negative signals generated by KIR2DL2 ligation.     

Retinoblastoma protein prevents staurosporine-induced cell death in a retinoblastoma-defective human glioma cell line.

OBJECTIVE: To investigate the mechanism of staurosporine-induced glioma cell death and cell cycle arrest using adenovirus-mediated gene transfection, as well as the function of retinoblastoma (Rb) and genetic instability induced by staurosporine. METHODS: Cell cycle regulation, cell death and nuclear abnormalities induced by staurosporine were examined using an adenovirus vector expressing Rb, p16 or p21 genes in human glioma cell lines. RESULTS: The Rb-defective SF-539 cell line was resistant to staurosporine compared with cell lines expressing intact Rb. SF-539 glioma cells exposed to staurosporine became multinucleated and then died. Multinucleation was prevented in SF-539 cells transfected with the Rb gene, thus decreasing the death rate of these cells. CONCLUSIONS: These results imply that enforced Rb expression protects cells from genomic instability induced by staurosporine regardless of its upstream molecular effects.     

Involvement of mitogen-activated protein kinase in peroxynitrite-induced cell death of human neuroblastoma SH-SY5Y cells

We have investigated the activation of mitogen-activated protein kinase (MAP kinase) in relation to cell death induced by peroxynitrite in human neuroblastoma SH-SY5Y cells. Exposure of the cells to peroxynitrite caused transient increase in MAP kinase activity, and resulted in cell death. PD98059, a selective inhibitor of MAP kinase kinase, reduced peroxynitrite-induced cell death. These results suggest that the activation of MAP kinase may be involved in cell death induced by peroxynitrite.     

Stimulus-dependent triggering or inhibition of cytotoxicity in human cytotoxic T lymphocytes by activators of protein kinase C.

The influence of activators of protein kinase C (PKC) on the delivery of the lethal hit by human cytotoxic T lymphocyte (CTL) clones was studied. In the absence of other signals, short-term incubation with two structurally unrelated activators of PKC, but not with a non-activating phorbolester, resulted in significant triggering of CTL, whereas overnight incubation with PKC activators led to reduction of cytotoxic activity. Furthermore, activation of PKC had an inhibitory effect on simultaneous triggering by anti-T3 monoclonal antibodies or by phytohaemagglutinin, but strongly enhanced the activating effect of anti-T11 antibodies. These results suggest that PKC is part of the cascade of signals transmitted within a CTL after triggering.     

DNA fragmentation and cell death mediated by T cell antigen receptor/CD3 complex on a leukemia T cell line

An anti-T cell receptor (TcR) monoclonal antibody (mAb), LC4, directed against a human leukemic T cell line, SUP-T13, caused DNA fragmentation ("apoptosis") and cell death upon binding to this cell line. Cross-linking of receptor molecules was necessary for this effect since F(ab')2, but not Fab', fragments of LC4 could induce cell death. Five anti-CD3 mAb tested also caused apoptosis, but only when they were presented on a solid phase. Interestingly, soluble anti-CD3 mAb induced calcium flux and had an additive effect on the calcium flux and interleukin 2 receptor expression induced by LC4, but these anti-CD3 mAb reversed the growth inhibition and apoptosis caused by LC4. The calcium ionophore A23187, but not the protein kinase C activator phorbol 12-myristate 13-acetate (PMA), also induced apoptosis, suggesting that protein kinase C activation alone does not cause apoptosis, although PMA is growth inhibitory. These results suggest that two distinct biological phenomena can accompany stimulation of the TcR/CD3 complex. In both cases, calcium flux and interleukin 2 receptor expression is induced, but only in one case is apoptosis and cell death seen. The signal initiating apoptosis can be selectively prevented by binding CD3 portion of the receptor in this cell line. This difference in signals mediated by the TcR/CD3 complex may be important in explaining the process of thymic selection, as well as in choosing anti-TcR mAb for therapeutic use.     

p38 MAP kinase mediates stress-induced leukotriene synthesis in a human B-lymphocyte cell line.

5-Lipoxygenase (5-LO), which catalyzes the first two steps in leukotriene biosynthesis, is a target for pharmacological treatment of inflammatory disorders. Previous studies have shown that B-lymphocytes express 5-LO. Here we demonstrate that several stimuli of cell stress such as osmotic shock (sorbitol, NaCl), oxidative stress (hydrogen peroxide, diamide), chemical stress sodium arsenite, and inflammatory cytokines enhanced cellular 5-LO activity in a B cell line (BL41-E95-A), when added simultaneously with ionophore plus arachidonate. It is interesting that sorbitol alone was sufficient for 5-LO product formation in the presence of exogenous arachidonic acid. These stimuli also activated p38 mitogen-activated protein (MAP) kinase and downstream MAP kinase-activated protein kinases in BL41-E95-A cells, which could phosphorylate 5-LO or heat shock protein 27 in vitro. The p38 MAP kinase inhibitor SB203580 abolished stress-induced leukotriene synthesis in B cells, without inhibition of 5-LO catalytic activity in cell-free systems. Our results indicate that p38 MAP kinase activation by cell stress is required for efficient leukotriene synthesis in B-lymphocytes.